Antimicrobial resistance is on the rise and it is estimated that more people will die from drug resistance infections than cancer by year 2050. As a result, there is an increasing interest in the discovery of new antimicrobial therapeutics. Although the majority of antibiotics were derived from soil microbes, current methods have been able to explore only approximately 1% of the soil microbiome. This is attributed to the difficulty in recapitulating environmental stimuli that facilitate the growth of these microbes in vitro. Cultivation-free approaches to identify putative antimicrobial-encoding gene clusters from soil metagenomes?with the goal of cloning and expressing such clusters in an heterologous host?are hampered by soil metagenome assembly of highly fragmented and chimeric contigs. Nevertheless, we hypothesize that these limitations can be harnessed in advantageous ways for the discovery of new antibiotics. We propose using an innovative high-throughput single-cell sequencing approach to predict full-length gene clusters from the assembled genomes. We will test this hypothesis in Aim 1 by developing a protocol for single-cell whole genome sequencing of soil microbes and identify putative full- length antimicrobial gene clusters. Our preliminary data using a controlled mock community demonstrates the feasibility of isolating, lysing, amplification, and sequencing of microbes in high-throughput. Furthermore, in Aim 2, we will use a microfluidics approach to screen soil microbes for the production of antibiotics that are active against multidrug resistant pathogens. We will also identify corresponding antibiotics-encoding gene cluster from its expression profile using an innovative approach that simultaneously characterizes both DNA and mRNA from a single cell. Altogether, the proposed approach will lay the groundwork for a novel method to discover new, soil- derived antibiotics-encoding gene cluster in high-throughput. This proposal is in line with my long term career goal of developing strategies to study microbial communities for the discovery of new therapeutics. Over the course of this award, I will be supported by my mentor Dr. Julia Oh, an expert in skin microbiome, and my co- mentor Dr. George Weinstock, a renowned microbiome and microbial genomics expert. In addition, I have assembled an advisory committee that includes Dr. Paul Robson, an expert in single cell genomics and transcriptomics, and Dr. Kim Lewis, a renowned expert in antimicrobial discovery from soil microorganisms. Collectively, this multi-disciplinary team will enable me to successfully execute the proposed experiments and advance my professional development as I transition to an independent position. Important research trainings proposed include soil microbiome handling and analyses, flow cytometry, microfluidics droplet merging techniques, RNA experimental protocols and analyses, and cloning of large-size DNA fragments. Professional development will include important elements such as grant writing and laboratory management. This project will be conducted at The Jackson Laboratory for Genomic Medicine, which offers full access to state-of-the art research laboratories and supporting facilities.